The present invention relates in general to methods and apparatus for rapidly and efficiently extracting nucleic acid from a biological sample and, in one embodiment, to a portable device for extracting nucleic acid, such as DNA or RNA, from a food sample in order to detect pathogens on food and crops. In addition to food microorganism detection, the present invention can be applied to detection of microorganisms in the medical and veterinary industries, surveillance of microorganisms in environmental applications, applications in homeland security and military field operations and routine microorganism screening in the pharmaceutical industry.
The need to detect microorganisms in food products has increased in recent years. Specifically, food products may be contaminated with pathogens either intentionally or unintentionally. Unintentional adulteration of crops pertains to environmental pathogens normally present and that contaminate the food product during normal food processing and handling procedures. Intentional adulteration of pathogens refers to a deliberate planting of pathogens on food or crops as would occur in an act of bioterrorism.
Often food products are tested for such microorganisms at the time that the food product is made. More often, the existence of such microorganisms does not become evident until after the food products have been sold at retail stores. Detecting a microorganism at such a late time generally results in a recall of vast amounts of food and/or a need to dispose of entire crops and whole food sources. Such recalls put an economic burden on food related businesses and pose a public health problem.
Most food manufacturers regularly analyze their food products. However, the detection systems in use today generally require the crop and/or food product to be tested at a laboratory facility that is remote from the processing plant or the field. Accordingly, the testing is time consuming and costly. Moreover, during the time of testing, contaminated food products may make their way into general commerce only to be recalled at a later date and after consumption.
Most conventional, manual methods of extracting nucleic acid are executed within a normal lab facility comprised of multiple steps. If the material to be processed is a solid matrix such as a food or tissue sample, the first step is to grind or otherwise mince the material. The process of grinding or mincing increases the surface area allowing the cell walls of microorganism embedded in the matrix to be exposed to the second step of cell lysis. The cell may be lysed by chemical treatment, boiling, enzymatic digestion of the cell wall, or by mechanical forces. Lysis releases the nucleic acid (i.e. DNA or RNA) from the cell and makes it available for manipulation. Following lysis, filtration may be employed to remove debris from the ground material. Subsequent organic extraction with solvents such as phenol and chloroform followed by centrifugation serves to separate proteins and lipids. In a third step, the DNA may be further purified via precipitation with salt and alcohol which also involves a centrifugation to pellet the DNA and allow removal of the supernatant. The DNA pellet may then be washed in an alcohol solution prior to resuspension in a tris-based buffer for analysis. DNA precipitation and washing serves to remove potential inhibitors that may affect downstream analytical approaches such as polymerase chain reaction (PCR). In addition to or in place of precipitation, the DNA may be purified over a column containing a solid phase material. After the DNA binds to the column, impurities can be removed by washing the column. The purified DNA can then be eluted off the column in a low salt aqueous solution for subsequent downstream analysis.
U.S. Pat. Nos. 6,120,985, 6,111,096, and 6,274,726, each issued to Laugharn, Jr. et al. describe methods for cell lysis of biological materials for purifying DNA for downstream applications. The methods include exposing biological cells to elevated pressures in a pressure chamber. In one patent the cells are cooled to subzero temperatures prior to placing them in the chamber. The sample chamber includes a filter and a solid phase material. The filter operates to remove cell debris from the sample. The solid phase material includes any one of silica gel, glass, plastic, membrane, resins, hydroxyapatite or tethered specific binding molecules or metals that are used to bind to nucleic acids in the sample. After the nucleic acids in the sample bind to the solid phase, the pressure in the chamber is increased to release the nucleic acids from the solid phase. The cartridge or chip with the solid phase material also contains electrodes to move biomolecules towards the solid phase for binding or towards a waste or collection reservoir. Modulating the pressure at the solid phase changes the binding of the biomolecule on the solid phase material. However, known devices for isolating and purifying DNA do not allow for integrated point-of-use analysis with solid food/tissue matrices.
The inventors of the present invention previously conceived combined mincing and cell lysis in one process prior to the purification step by putting a whole food/tissue sample in the lysis solution and then putting the solution in the grinding/mincing chamber of a lysis tube for food/tissue comminution. The tube includes an upper sample preparation chamber having at least one mincing disk to mince the sample, a filter to filter particulate matter from the extract, and a third chamber having a binding material configured to bind to the DNA of the extract. The whole food/tissue sample is forced through pores of lysis disks to mince the food/tissue sample and then, the minced food/tissue sample is filtered prior to the purification step.
The present invention aims to provide an alternative system and method for extracting nucleic acid from a nucleic acid-containing sample.